Anti-human folate receptor beta antibodies and methods of use

ABSTRACT

Human anti-human folate receptor beta antibodies and antigen-binding fragments thereof are described, as well as methods of using such antibodies and fragments to treat inflammatory disorders or cancers expressing cell surface FRβ.

SEQUENCE LISTING

A sequence listing in electronic (ASCII text file) format is filed withthis application and incorporated herein by reference. The name of theASCII text file is “AttachE_Sequence_Listing.txt”; the file was createdon Apr. 8, 2016; the size of the file is 17 KB.

TECHNICAL FIELD

This invention relates to anti-folate receptor beta (FRβ) antibodies,and more particularly to human monoclonal antibodies, or antigen-bindingfragments thereof, that specifically bind human FRβ and methods of usingsuch antibodies or antigen binding fragments thereof to reduce thenumber of FRβ positive cells in subjects.

BACKGROUND

Folic acid is a vitamin required for the synthesis of nucleotide basesand is essential for the proliferation of all cells. Folates also arerequired for production of S-adenosylmethionine, the common substrateused in methylation of DNA, histones, G proteins, and many metabolicbuilding blocks (see Kim, J Nutr 135:2703-2709 (2005); Loenen, BiochemSoc Trans 34:330-333 (2006)). Almost all cells take in folic acid viathe reduced folate carrier or proton coupled folate transporter (seeMatherly and Goldman, Vitam Horm 66:403-456 (2003)). Some cells,however, also express a folate receptor (FR) that binds folic acid˜100,000 times tighter than the aforementioned transporters, and carriesbound folates into cells by receptor-mediated endocytosis (seeNakashima-Matsushita et al., Arth Rheum 42:1609-1616 (1999); Turk etal., Arthritis Rheumatoid 46:1947-1955 (2002)). There are four membersof the FR family: FRα, FRβ, FRγ and FRδ (see Elnakat and Ratnam, AdvDrug Deliv Rev 56:1067-1084 (2004)). Different isoforms of the FR areused by certain cancer cells, activated macrophages, and the proximaltubule cells of the kidney to capture folates from their environment(see e.g., Nakashima-Matsushita et al. 1999, supra; and Turk et al.2002, supra). A need exists for reagents and methods for differentialtargeting of the folate receptors for treatment of disease.

SUMMARY

This document is based on, inter alia, the identification of a humanmonoclonal antibody that specifically binds FRβ. Such an antibody or anantigen binding fragment thereof can be used to reduce the number of FRβpositive cells in a subject, and is useful for treating inflammatorydisorders and cancers that express cell surface FRβ. The antibodydescribed herein is particularly useful for treating human subjects asit is fully human, with reduced risk for inducing immune responses andside effects compared with murine, chimeric, or humanized antibodies.

In one aspect, this document features an isolated human monoclonalantibody, or an antigen-binding fragment thereof (e.g., Fab, a F(ab′)₂fragment, or a single chain antibody fragment (scFv)) that specificallybinds human folate receptor beta (FRβ). The antibody or fragment caninclude a heavy chain variable region (V_(H)) complementaritydetermining region (CDR) 1 comprising the amino acid sequence set forthin SEQ ID NO:1; a V_(H) CDR2 comprising the amino acid sequence setforth in SEQ ID NO:2; a V_(H) CDR3 comprising the amino acid sequenceset forth in SEQ ID NO:3; a light chain variable region (V_(L)) CDR1comprising the amino acid sequence set forth in SEQ ID NO:4; a V_(L)CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5; and aV_(L) CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6.The antibody or fragment can include one or more framework regions inSEQ ID NO: 7 and SEQ ID NO:8. The antibody or fragment can have theamino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO:11. Theantibody or fragment can have the amino acid sequence set forth in SEQID NO:7 or SEQ ID NO:8. The antibody or fragment can bind to cellsurface FRβ. The antibody can be an IgG₁ antibody. The antibody orfragment can induce antibody-dependent cell-mediated cytotoxicity (ADCC)of FRβ expressing target cells. In some embodiments, the antibody orfragment is de-fucosylated. In some embodiments, the antibody orfragment does not detectably bind to human folate receptor gamma (FRγ)or human folate receptor delta (FRδ).

In another aspect, this document features an isolated human monoclonalantibody, or an antigen-binding fragment thereof (e.g., Fab, a F(ab′)₂fragment, or a scFv) that specifically binds human FRβ. The antibody orfragment can include a V_(H) CDR 1 comprising the amino acid sequenceset forth in SEQ ID NO:1; a V_(H) CDR2 comprising the amino acidsequence set forth in SEQ ID NO:2; a V_(H) CDR3 comprising the aminoacid sequence set forth in SEQ ID NO:3; a V_(L) CDR1 comprising theamino acid sequence set forth in SEQ ID NO:14; a V_(L) CDR2 comprisingthe amino acid sequence set forth in SEQ ID NO:15; and a V_(L) CDR3comprising the amino acid sequence set forth in SEQ ID NO:16. Theantibody or fragment can include one or more framework regions in SEQ IDNO: 7 and SEQ ID NO: 17. The antibody or fragment can have the aminoacid sequence set forth in SEQ ID NO:10 or SEQ ID NO:18. The antibody orfragment can have the amino acid sequence set forth in SEQ ID NO:7 orSEQ ID NO:17. The antibody or fragment can bind to cell surface FRβ. Theantibody can be an IgG₁ antibody. The antibody or fragment can induceADCC of FRβ expressing target cells. In some embodiments, the antibodyor fragment is de-fucosylated. In some embodiments, the antibody orfragment does not detectably bind to human FRγ or human FRδ.

This document also features an isolated human monoclonal antibody, or anantigen-binding fragment thereof (e.g., Fab, a F(ab′)₂ fragment, or ascFv) that specifically binds human FRβ. The antibody or fragmentcontains a V_(H) CDR 1 comprising the amino acid sequence set forth inSEQ ID NO:1; a V_(H) CDR2 comprising the amino acid sequence set forthin SEQ ID NO:2; and a V_(H) CDR3 comprising the amino acid sequence setforth in SEQ ID NO:3. The antibody or fragment has one or moreproperties selected from the group consisting of: (a) the antibody orfragment does not detectably bind to human folate receptor alpha (FRα);(b) the antibody or fragment binds to human macrophages but not to mousemacrophages; (c) the antibody or fragment has a binding affinity (EC₅₀)of 20 nM; (d) the antibody or fragment has a dissociation constant (Kd)of 6.39 nM; and (e) the antibody mediates ADCC of FRβ-expressing targetcells. In some embodiments, the antibody or fragment does not detectablybind to human FRγ or human FRδ.

This document also features an isolated heavy chain of an antibody, orantigen binding fragment of an antibody, that specifically binds humanFRβ. The isolated heavy chain comprises a V_(H) CDR 1 comprising theamino acid sequence set forth in SEQ ID NO:1; a V_(H) CDR2 comprisingthe amino acid sequence set forth in SEQ ID NO:2; and a V_(H) CDR3comprising the amino acid sequence set forth in SEQ ID NO:3, wherein theantibody or fragment has one or more properties selected from the groupconsisting of: (a) the antibody or fragment does not detectably bind tohuman FRα; (b) the antibody or fragment binds to human macrophages butnot to mouse macrophages; (c) the antibody or fragment has a bindingaffinity (EC₅₀) of 20 nM; (d) the antibody or fragment has adissociation constant (Kd) of 6.393 nM; and (e) the ADCC ofFRβ-expressing target cells. In some embodiments, the antibody orfragment does not detectably bind to human FRγ or human FRδ.

An antibody or fragment described herein can be conjugated with apharmaceutical agent (e.g., a chemotherapeutic) or a liposome (e.g., aliposome including a pharmaceutical agent), linked to a toxin (e.g.,covalently linked to the toxin) or a detectable moiety. The detectablemoiety can be selected from the group consisting of a fluorescentmoiety, a luminescent moiety, a radioactive moiety, a CT contrast agent,an MRI contrast agent, and biotin.

This document also features compositions including any of the antibodiesor fragments described herein and a pharmaceutically acceptable carrier,and methods of using such compositions. For example, a composition canbe used in a method of treating a patient having an inflammatorydisorder (e.g., atherosclerosis, ischemia/reperfusion injury,transplantation rejection, vasculitis, inflammatory osteoarthritis,glomerulonephritis, restenosis, systemic sclerosis, fibromyalgia,sarcoidosis, or an autoimmune disease such as rheumatoid arthritis,systemic lupus erythematosus (SLE), ulcerative colitis, psoriasis, Type1 diabetes, Crohn's disease, multiple sclerosis, and Sjogren's disease).The method can include administering to the patient an amount of acomposition effective to reduce the number of FRβ positive macrophagesand monocytes in the patient.

A composition described herein also can be used in a method of treatinga patient having a cancer expressing cell-surface FRβ. The method caninclude administering to the patient, an amount of the compositioneffective to reduce the number of FRβ expressing cancer cells in thepatient. The cancer can be a myeloid cancer (e.g., acute myeloidleukemia or chronic myeloid leukemia), multiple myeloma, or a solid FRβexpressing cancer (e.g., a non-epithelial malignancy).

This document also features a method of depleting activated macrophagesfrom a human subject. The method includes administering to the subject ahuman monoclonal antibody, or antigen-binding fragment thereof, thatspecifically binds human FRβ, in an amount effective to reduce thenumber of activated macrophages in the subject.

In any of the methods described herein, the antibody or fragment thereofcan induce ADCC of FRβ expressing target cells. In some embodiments, theantibody or fragment is de-fucosylated.

In any of the methods described herein, the antibody or fragment caninduce opsonization-mediated clearance of FRβ expressing target cells.

In any of the methods described herein, the antibody or fragment caninduce complement-mediated lysis of FRβ expressing target cells.

This document also features a nucleic acid that includes first andsecond segments, the first segment encoding a scFv fragment of a humanmonoclonal antibody that specifically binds human FRβ, and the secondsegment encoding at least the intracellular domain of a T cell signalingpolypeptide. The T cell signaling polypeptide is a CD3 zeta chain orFcRγ chain. The nucleic acid further can include a third segmentencoding at least the intracellular domain of a costimulatorypolypeptide (e.g., a costimulatory polypeptide selected from the groupconsisting of CD28, CD40L, CD134, CD137, and PD-1). In addition, thisdocument features cells that include any such nucleic acids. The cellscan be T cells (e.g., cytotoxic T cells, CD8+ T cells, or CD4+ T cells).The T cells can produce at least one cytokine or lymphokine (e.g.,interferon-γ (IFN-γ)).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a depiction of the amino acid sequence of the full-length,unprocessed human FRβ protein (SEQ ID NO:9). A fragment of thefull-length protein containing residues 22 to 236 was used to producerecombinant FRβ.

FIG. 2A is a depiction of the amino acid sequence of the m909 Fab heavychain sequence (SEQ ID NO:7) and FIG. 2B is a depiction of the aminoacid sequence of the m909 Fab light chain lambda sequence (SEQ IDNO:17). In each of the sequences, the framework regions are italicized,the CDRs are underlined, and the constant region is in lowercase,non-italicized text. Framework regions 1-4 (FR1-FR4) are numberedconsecutively from the N-terminus, with FR1 being the most N-terminalsequence. CDRs 1-3 also are numbered consecutively from the N-terminus,with CDR1 being the most N-terminal.

FIG. 3 is a graph showing the binding of m909 IgG to FRβ.

FIG. 4 is a one-dimensional fluorescent flow cytometry (FFC) histogramshowing that bivalency allows binding of scFv m909 to FRβ positivecells.

FIG. 5 is a one-dimensional FFC histogram showing that scFv m909 doesnot bind to FRβ negative cells.

FIGS. 6A and 6B depict isoform-specific binding of FITC-human anti-humanFRβ mAb to FRβ-positive/negative cell lines. FIG. 6A contains fourone-dimensional FFC histograms of control FITC-isotype IgG1 binding toCHO-hFRβ (a) or FITC-human anti-human FRβ IgG1 binding to CHO-hFRβ cells(b), KB cells (c), or CHO-K1 cells. FIG. 6B is a line graph showing theconcentration of fluorescent intensity vs. human anti-human FRβ IgG1.

FIG. 7 is a bar graph showing the percent lysis of preB L1.2FRβ-positive cells at various concentrations of IgG₁ m909 or controlIgG₁.

FIG. 8 contains a bar graph of the percent lysis of CHO-FRβ cells (leftpanel) and a bar graph of the percent lysis of CHO-K1 cells (rightpanel).

FIG. 9A contains representative 2-dimensional and 1-dimensional FFCanalyses of FITC-anti-FRβ m909 IgG1 binding to bacteria-recruitedperitoneal FRβ+ macrophages. In a), FRβ+ macrophages were stained withfolate conjugated to FITC. In b), FRβ+ macrophages were stained withFITC conjugated to human anti-human FRβ m909 IgG1. The percentage ofFRβ+ cells within each gate shown in the left hand two panels(2-dimensional FFC histograms) are shown in the right hand two panels(1-dimensional FFC histograms).

FIG. 9B contains representative 2-dimensional and one-dimensional FFCanalyses of FITC-anti-FRβ m909 IgG1 binding to the FRβ+CD14^(High) CD16⁻subset of human peripheral blood monocytes. The populations of cellsgated in the left hand 2-dimensional histograms were subjected toanalysis using fluorochrome-conjugated anti-CD16 and anti-CD14antibodies (middle two 2-dimensional FFC histograms) and the percentageof FRβ+ cells in the gated populations in the latter histograms weredetermined (right hand 1-dimensional FFC histograms).

DETAILED DESCRIPTION

In general, this document provides human anti-human FRβ monoclonalantibodies or antigen-binding fragments thereof, as well as methods ofusing such antibodies or fragments thereof to treat, detect, or monitoran inflammatory disorder or a cancer expressing FRβ in a patient (e.g.,a human patient). The term “FRβ” as used herein refers to human folatereceptor beta. The amino acid sequence of human FRβ can be found in FIG.1 (SEQ ID NO:9) and in GenBank under Accession No. NP_001107007. FRβ isa differential marker on the surface of myelomonocytic lineage cells. Innormal tissues, FRβ is expressed in placenta, myelomonocytic lineagecells (e.g., monocytes and macrophages), and mature neutrophils. FRβ,however, does not bind folic acid on quiescent macrophages until themyeloid cell becomes activated. FRβ is consistently detected in multiplemyeloma cells, chronic myeloid leukemia (CML) cells, and in 70% of acutemyeloid leukemia (AML) cells. See, for example, Nakashima-Matsushita etal., 1999, supra; and Ross et al., Contrast Media Mol Imaging 2:72-81(1999). FRβ also has been detected by RT-PCR in solid tumors (e.g.,carcinomas from colon, kidney, breast, ovary, uterus, or lung; squamouscell carcinomas of the head and neck; and malignancies of non-epithelialorigin such as sarcomas, lymphomas, fibrous histiocytomas, ovariangranulosa cell tumor, astrocytoma, meningiomas, and Wilms' tumor). See,for example, Ross et al., Cancer, 73:2432-43 (1994).

In monocytes and macrophage-lineage cells, FRβ expression is increasedupon activation. For example, FRβ is expressed and functional insynovial macrophages in rheumatoid arthritis patients. Furthermore,γ-scintigraphy images of patients with a variety of inflammatorydisorders (e.g., rheumatoid arthritis, Crohn's disease, ischemic boweldisease, Sjogren's syndrome, localized infections, atherosclerosis, andorgan transplant rejection) show uptake of a folate-targeted ^(99m)Tcimaging agent (EC20) at sites of inflammation (see Low et al., Acc ChemRes. 41(1):120-9 (2008); Matteson et al., Clin Exp Rheumatol 27:253-259(2009); and Ayala-Lopez et al., J Nuc Med, 51:768-774 (2010)). Thesefindings indicate expression of FRβ in activated macrophages andmonocytes.

The antibodies and antigen binding fragments described hereinspecifically bind FRβ, and do not detectably bind FRI In someembodiments, the antibodies and antigen binding fragments describedherein also do not detectably bind FRK and/or FRΛ. FRα is expressed onthe apical surfaces of a few epithelial cell types (primarily proximaltubules of the kidneys and alveolar epithelial cells of the lungs) andis upregulated on a variety of epithelial-derived tumors (see Weitman etal., Cancer Res 52:6708-6711 (1992); Salazar and Ratnam, CancerMestastasis Rev 26:141-152 (2007); and Leamon and Jackman, Vitam Horm79:203-233 (2008)). FRγ is rarely expressed and difficult to detect invivo. FRδ is expressed on regulatory T cells, where it exhibits onlyvery low affinity for folic acid. As such, the antibodies and antigenbinding fragments described herein, which specifically bind FRβ, can beused for depleting activated macrophages, treating inflammatorydisorders, and treating cancers that express FRβ.

Human Anti-Human FR/3 Antibodies

This document features fully human monoclonal antibodies (mAb), orantigen-binding fragments thereof, that specifically bind human FRβ. Asused herein, the term “antibody” refers to a protein that generallycomprises heavy chain polypeptides and light chain polypeptides. IgG,IgD, and IgE antibodies comprise two heavy chain polypeptides and twolight chain polypeptides. IgA antibodies comprise two or four of eachchain and IgM generally comprise 10 of each chain. Single domainantibodies having one heavy chain and one light chain and heavy chainantibodies devoid of light chains are also contemplated. A givenantibody comprises one of five types of heavy chains, called alpha (1 or2), delta, epsilon, gamma (1, 2, 3, or 4), and mu, the categorization ofwhich is based on the amino acid sequence of the heavy chain constantregion. These different types of heavy chains give rise to five classesof antibodies, IgA (including IgA1 and IgA2), IgD, IgE, IgG (IgG1, IgG2,IgG3 and IgG4) and IgM, respectively.

In an IgG antibody, the heavy chain includes a “hinge region”, “C_(H)1region”, “C_(H)2 region”, “C_(H)3 region” each of which refers to adifferent region of the heavy chain constant region. The hinge region islocated between the C_(H)1 region and C_(H)2 region, and links the Fabregion to the Fc region. Thus, N terminal to C terminal, the regions arein the order C_(H)1, hinge, C_(H)2, and C_(H)3. In the μ heavy chainconstant regions of IgM antibodies, there is an extra C-terminal region,the C_(H)4 region. The antibodies and fragments of this document cancontain all or some of the regions of the heavy chain constant regions.Thus, they can contain all or part or the hinge region, and/or all orpart of the C_(H)1 region, and/or all or part of the C_(H)2 region,and/or all or part of the C_(H)3 region, and/or all or part of theC_(H)4 region. Moreover, any of these regions, or parts of regions, canin any particular antibody or fragment, be derived from one, two, three,four, five, six, seven, eight, or nine different constant region classesor subclasses (K1, 2, 3, or 4, I1 or 2, M, μ, and Λ).

Antibodies and antigen binding fragments described herein can be of anyclass and have the corresponding heavy chain constant region. A givenantibody also comprises one of two types of light chains, called kappaor lambda, the categorization of which is based on the amino acidsequence of the light chain constant regions. Antibodies and antigenbinding fragments described herein can have either class of light chain.

Fully human monoclonal antibodies that specifically bind to FRβ can beproduced, e.g., using in vitro-primed human splenocytes as described byBoerner et al., J. Immunol., 147, 86-95 (1991), or repertoire cloning asdescribed by Persson et al., Proc. Nat. Acad. Sci. USA, 88: 2432-2436(1991); Huang and Stollar, J. Immunol. Methods 141, 227-236 (1991); orU.S. Pat. No. 5,798,230. Large nonimmunized or immunized human phagedisplay libraries also can be used to isolate high affinity antibodiesusing standard phage technology (see, e.g., Hoogenboom et al.,Immunotechnology 4:1-20 (1998); Hoogenboom et al., Immunol Today 2:371-8(2000); and U.S. Patent Publication No. 2003-0232333). In addition, aTransChromo mouse carrying human immunoglobulin loci, including all thesubclasses of IgGs (IgG1-G4), can be used to prepare human monoclonalantibodies. See, Ischida et al., Cloning Stem Cells 4(1):91-102 (2002).

“Antigen binding fragment” of an antibody as the term is used hereinrefers to an antigen binding molecule that is not a complete antibody asdefined above, but that still retains at least one antigen binding site.Thus, in one embodiment, an antigen binding fragment is a fragment of anantibody that binds to FRβ. Antibody fragments often include a cleavedportion of a whole antibody, although the term is not limited to suchcleaved fragments. Antigen binding fragments can include, for example, aFab, F(ab′)₂, Fv, and single chain Fv (scFv) fragment. An scFv fragmentis a single polypeptide chain that includes both the heavy and lightchain variable regions of the antibody from which the scFv is derived.Other suitable antibodies or antigen binding fragments include linearantibodies, multispecific antibody fragments such as bispecific,trispecific, and multispecific antibodies (e.g., diabodies (PoljakStructure 2(12):1121-1123 (1994); Hudson et al., J. Immunol. Methods23(1-2):177-189 (1994)), triabodies, tetrabodies), minibodies, chelatingrecombinant antibodies, intrabodies (Huston et al., Hum. Antibodies10(3-4):127-142 (2001); Wheeler et al., Mol. Ther. 8(3):355-366 (2003);Stocks Drug Discov. Today 9(22): 960-966 (2004)), nanobodies, smallmodular immunopharmaceuticals (SMIP), binding-domain immunoglobulinfusion proteins, camelid antibodies, camelized antibodies, and V_(HH)containing antibodies.

A human anti-human FRβ mAb or antigen-binding fragment thereof describedherein specifically binds to human FRβ, but does not detectably bind toFRI. In some embodiments, the anti-human FRβ mAb or antigen-bindingfragment thereof also does not detectably bind to FRK and/or FRΛ.Binding specificity can be assessed as described herein. For example,binding specificity of an antibody or antigen binding fragment thereofcan be assessed using recombinantly produced FR or cells expressing aparticular FR on their cell surface and techniques such as enzyme linkedimmunosorbent assay (ELISA) or flow cytometry. As described herein,human anti-human FRβ mAb can bind cell surface FRβ, and antigen bindingfragments of human anti-human FRβ mAb (e.g., Fab or scFv) can bind cellsurface FRβ mAb when bivalent. Human anti-human FRβ mAb can bind tohuman macrophages but not to mouse macrophages. As described herein, inFab format, m909 bound to recombinant FRβ with high (nM) affinity(EC₅₀=20 nM). In an IgG1 format, m909 has high (subnanomolar) avidity invitro and specifically binds to cell surface FRβ. As described inExample 3, the human anti-human FRβ mAb has a dissociation constant (Kd)of 6.393 nM.

In some embodiments, an antibody or antigen binding fragment thereofdescribed herein contains a heavy chain variable region (V_(H))complementarity determining region (CDR) 1 having the amino acidsequence set forth in SEQ ID NO:1 [GYTFTSYA], a V_(H) CDR2 having theamino acid sequence set forth in SEQ ID NO:2 [KYSQKFQ]; and a V_(H) CDR3having the amino acid sequence set forth in SEQ ID NO:3 [ARDISYGSFDYW].Alternatively, or in addition, the antibody or antigen binding fragmentthereof described herein contains a light chain variable region (V_(L))CDR1 having the amino acid sequence set forth in SEQ ID NO:4 [NLRSYY]; aV_(L) CDR2 having the amino acid sequence set forth in SEQ ID NO:5[GKN]; and a V_(L) CDR3 having the amino acid sequence set forth in SEQID NO:6 [HSRKSRGNHLLF].

In some embodiments, the antibody or fragment described herein caninclude one or more framework regions italicized in the following aminoacid sequence:

(SEQ ID NO: 7 [EVQLVQSGAEVKKPGASVKVSCKAS GYTFTSYA MHWVRQAPGQRLEWMGWINAGNGNT KYSQKFQ GRVTITRDTSASTAYMELSSLRSEDTAVYYC ARD ISYGSFDYWGQGTLVTVSSastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdktsgqag],heavy chain sequence; constant region in lower class letters)).Alternatively, or in addition, the antibody or fragment described hereincan include one or more framework regions italicized in the followingamino acid sequence:

(SEQ ID NO: 8 [SSELTQDPAVSVALGQTVRITCQGD NLRSYY ASWYRQKSGQAPVLVIY G KNNRPSGIPDRFSGSSSGNTASLTITAAQAEDEADYYC HSRKSRGNHLLFGGGTKLTVLgqpkaapsvtlfppsseelqankatlvclisdfypgavtvakadsspvkagvetttpskqsnnkyaassylsltpeqwkshrsyscqvthe gstvektvaptecs],lambda light chain sequence; constant region in lower case letters).

In some embodiments, the antibody or antigen binding fragment thereofcomprises the heavy chain variable region (V_(H)) amino acid sequenceset forth in SEQ ID NO:10

[EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARD ISYGSFDYWGQGTLVTVSS].Alternatively, or in addition, the antibody or antigen binding fragmentthereof comprises the light chain variable region amino (V₁) amino acidsequence set forth in SEQ ID NO:11

[SSELTQDPAVSVALGQTVRITCQGDNLRSYYASWYRQKSGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITAAQAEDEADYYCHSRKSRGNHLLF GGGTKLTVL].In some embodiments, an antibody or fragment thereof comprising suchsequences is designated m923 and specifically binds to human FRβ.

In some embodiments, the antibody or fragment described herein caninclude the following heavy chain amino acid sequence:

(SEQ ID NO: 7) [EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISYGSFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTSGQAG].Alternatively, or in addition, the antibody or fragment described hereincan include the following light chain amino acid sequence:

(SEQ ID NO: 8) [SSELTQDPAVSVALGQTVRITCQGDNLRSYYASWYRQKSGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITAAQAEDEADYYCHSRKSRGNHLLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE GSTVEKTVAPTECS].

In some embodiments, the antibody or antigen binding fragment thereofdescribed herein contains a heavy chain variable region (V_(H))complementarity determining region (CDR) 1 having the amino acidsequence set forth in SEQ ID NO:1 [GYTFTSYA], a V_(H) CDR2 having theamino acid sequence set forth in SEQ ID NO:2 [KYSQKFQ]; and a V_(H) CDR3having the amino acid sequence set forth in SEQ ID NO:3 [ARDISYGSFDYW].Alternatively, or in addition, the antibody or antigen binding fragmentthereof described herein contains a light chain variable region (V_(L))CDR1 having the amino acid sequence set forth in SEQ ID NO:14 [SLRSNY];a V_(L) CDR2 having the amino acid sequence set forth in SEQ ID NO:15[GQN]; and a V_(L) CDR3 having the amino acid sequence set forth in SEQID NO:16 [DSRVSTGNHVVF].

In some embodiments, an antibody or fragment described herein caninclude one or more framework regions italicized in the following aminoacid sequence:

(SEQ ID NO: 7 [EVQLVQSGAEVKKPGASVKVSCKAS GYTFTSYA MHWVRQAPGQRLEWMGWINAGNGNT KYSQKFQ GRVTITRDTSASTAYMELSSLRSEDTAVYYC ARD ISYGSFDYWGQGTLVTVSSastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdktsgqag],heavy chain sequence; constant region in lower case letters).Alternatively, or in addition, the antibody or fragment described hereincan include one or more framework regions italicized in the followingamino acid sequence:

(SEQ ID NO: 17 [SSELTQDPAVSVALGQTVRITCQGD SLRSNY ANWYQQKPGQAPVLVIY G QNNRPSGIPDRFSGSSSGNTASLTITGAQAADEADYYC DSRVSTGNHVVFGGGTKLTVLgqpkaapsvtlfppsseelqankatlvclisdfypgavtvakadsspvkagvetttpskqsnnkyaassylsltpeqwkshrsyscqvthe gstvektvaptecs],lambda light chain sequence; constant region in lower case letters).

In some embodiments, the antibody or antigen binding fragment thereofcomprises the V_(H) amino acid sequence set forth in SEQ ID NO:10

[EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARD ISYGSFDYWGQGTLVTVSS].Alternatively, or in addition, the antibody or antigen binding fragmentthereof comprises the V_(L) amino acid sequence set forth in SEQ IDNO:18

[SSELTQDPAVSVALGQTVRITCQGDSLRSNYANWYQQKPGQAPVLVIYGQNNRPSGIPDRFSGSSSGNTASLTITGAQAADEADYYCDSRVSTGNHVVF GGGTKLTVL].In some embodiments, the antibody or antigen binding fragment thereof isdesignated m909.

In some embodiments, an antibody or fragment described herein caninclude the following heavy chain amino acid sequence:

(SEQ ID NO: 7) [EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISYGSFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTSGQAG].Alternatively, or in addition, the antibody or fragment described hereincan include the following light chain amino acid sequence:

(SEQ ID NO: 17) [SSELTQDPAVSVALGQTVRITCQGDSLRSNYANWYQQKPGQAPVLVIYGQNNRPSGIPDRFSGSSSGNTASLTITGAQAADEADYYCDSRVSTGNHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT HEGSTVEKTVAPTECS].

It is understood that antibodies and antigen binding fragments describedabove, whether specified in terms of heavy and light chain components,or heavy or light chain components, can have any of the functionalproperties and activities described herein.

In some embodiments, the full length heavy or light chain, variableregion, CDR, framework region, or constant region of an antibody orantigen binding fragment described herein can have at least 80% sequenceidentity (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequenceidentity) to the corresponding amino acid sequence set forth in any oneof SEQ ID NOs:1-8, 10, 11, and 14-18. The percent identity between aparticular amino acid sequence and the amino acid sequence set forth inany one of SEQ ID NOs:1-8, 10, 11, and 14-18 can be determined asfollows. First, the amino acid sequences are aligned using the BLAST 2Sequences (Bl2seq) program from the stand-alone version of BLASTZcontaining BLASTP version 2.0.14. This stand-alone version of BLASTZ canbe obtained from Fish & Richardson's web site (e.g., www.fr.com/blast/)or the U.S. government's National Center for Biotechnology Informationweb site (www.ncbi.nlm.nih.gov). Instructions explaining how to use theBl2seq program can be found in the readme file accompanying BLASTZ.Bl2seq performs a comparison between two amino acid sequences using theBLASTP algorithm. To compare two amino acid sequences, the options ofBl2seq are set as follows: -i is set to a file containing the firstamino acid sequence to be compared (e.g., C:\seq1.txt); -j is set to afile containing the second amino acid sequence to be compared (e.g.,C:\seq2.txt); -p is set to blastp; -o is set to any desired file name(e.g., C:\output.txt); and all other options are left at their defaultsetting. For example, the following command can be used to generate anoutput file containing a comparison between two amino acid sequences:C:\Bl2seq c:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If thetwo compared sequences share homology, then the designated output filewill present those regions of homology as aligned sequences. If the twocompared sequences do not share homology, then the designated outputfile will not present aligned sequences. Similar procedures can befollowed for nucleic acid sequences except that blastn is used.

Once aligned, the number of matches is determined by counting the numberof positions where an identical amino acid residue is presented in bothsequences. The percent identity is determined by dividing the number ofmatches by the length of the amino acid sequence in SEQ ID NOs:1-8, 10,11, or 14-18, followed by multiplying the resulting value by 100.

It is noted that the percent identity value is rounded to the nearesttenth. For example, 78.11, 78.12, 78.13, and 78.14 is rounded down to78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2.It also is noted that the length value will always be an integer.

It will be appreciated that a number of nucleic acids can encode theamino acid sequences set forth in SEQ ID NOs:1-8, 10, 11, and 14-18. Thedegeneracy of the genetic code is well known to the art; i.e., for manyamino acids, there is more than one nucleotide triplet that serves asthe codon for the amino acid. The nucleotide sequence encoding the aminoacid sequence of SEQ ID NO:7 is set forth in SEQ ID NO: 19. Thenucleotide sequence encoding the amino acid sequence of SEQ ID NO:8 isset forth in SEQ ID NO: 20. The nucleotide sequence encoding the aminoacid sequence of SEQ ID NO: 17 is set forth in SEQ ID NO:21. In someembodiments, a heavy or light chain of an antibody is encoded by anucleic acid having at least 80% sequence identity (e.g., at least 85%,90%, 95%, 97%, 98%, 99%, or 100% sequence identity) to the nucleotidesequences set forth in SEQ ID NOs. 19-21. Sequence identity iscalculated as described above for protein sequences except that blastnis used.

In some embodiments, an antibody or antigen binding fragment thereofdescribed herein induces antibody-dependent cell-mediated cytotoxicity(ADCC) of FRβ expressing target cells. Suitable antigen bindingfragments that induce ADCC have a functional Fc region, i.e., an Fcregion that can bind to an Fc receptor on an ADCC effector cell. Theantibodies and antigen binding fragments thereof can bind to IgG Fcbinding receptors (FcγR) receptors I, II and III (CD64, CD32, and CD16)in order to mediate the functional activities described herein. In someembodiments, binding to a FcKR (e.g., FcKR III) can be enhanced byremoving fucose residues from and/or by increasing galactosylation ofthe glycan present on the Fc portion of an IgG₁. See, for example,Houdes et al., Molecular & Cellular Proteomics 9:1716-1728 (2010);Kubota et al., Cancer Sci., 100: 1566-1572 (2009); Malphettes et al.,Biotechnol. Bioeng., 106:774-783 (2010); and Raju, Curr. Opin. Immunol.,20:471-478 (2008). De-fucosylated antibodies can be produced using, forexample, cells with reduced expression of the GDP-4,6-dehydratase gene(e.g., from a mutation such as that in Chinese hamster ovary (CHO) Lec13cells or from a small interfering RNA against the GDP-4,6-dehydratasegene), cells in which the I-1,6-fucosyltransferase (FUT8) has beenknocked out or expression reduced (e.g., using a small interfering RNA(siRNA) against the FUT8 gene), cells co-expressingβ-1,4-N-acetylglucosaminyltransferase III (GnT-III) and Golgi Imannosidase II (ManII), or cells expressingGDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD). See, Ishiguro et al.,Cancer Sci., pages 1-7, July, 2010; and von Horsten, Glycobiology,published online Jul. 23, 2010. FcKR binding also can be enhanced bymutating relevant amino acids in the heavy chain constant regionscomprising the Fc region (e.g., the hinge region, the C_(H)2 region, orthe C_(H)3 region). See, for example, Natsume et al., Drug Des. Develop.Ther., 3:7-16 (2009). In some embodiments, an antibody or fragmentthereof can induce opsonization-mediated clearance and/or inducecomplement-mediated lysis of FRβ expressing target cells. ADCC can beassessed in vitro using a lactate dehydrogenase (LDH) release assay orchromium-51 release assay.

This document also provides antibodies that bind to particular epitopesof FRβ that contain, for example, at least three amino acids of humanFRβ (FIG. 1, SEQ ID NO: 9). For example, an epitope can contain 3 to 30such as 5 to 25, 7 to 23, 10 to 20, or 13 to 18 amino acids of humanFRβ). For example, an epitope can contain 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 amino acids of human FRβ. The epitope can be in the N-terminalhalf (i.e., residues 1 to 127 of SEQ ID NO: 9) or the C-terminal half ofhuman FRβ (i.e., residues 128 to 255 of SEQ ID NO:9).

An antibody or antigen binding fragment described herein can beconjugated or linked, either covalently or noncovalently, to a varietyof molecules, including pharmaceutical agents, liposomes,oligonucleotides (e.g., small interfering RNA (siRNA)), toxins,detectable moieties, or biological molecules (e.g., a cytokine such asan interleukin (IL) 2, IL4, IL12, 13 or 15; interferon (IFN) I, IFN β,or IFN K) using methods known in the art. Conjugating or linking suchmolecules to an antibody or antigen binding fragment described hereinallows the molecule to be targeted specifically to FRβ. As such,molecules can be delivered to the desired site while minimizingtoxicity.

For example, an antibody or antigen binding fragment can be conjugatedwith a pharmaceutical agent such as a chemotherapeutic (e.g., cisplatin,carboplatin, procarbazine, mechlorethamine, cyclophosphamide,camptothecin, adriamycin, ifosfamide, melphalan, chlorambucil, bisulfan,nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin,plicomycin, mitomycin, etoposide, verampil, podophyllotoxin, tamoxifen,taxol, transplatinum, 5-flurouracil, vincristin, vinblastin,methotrexate, or an analog of any of the aforementioned). An antibody orantigen binding fragment also can be conjugated with ananti-inflammatory agent such as a glucocorticoid, nonsteroidalanti-inflammatory agent, phosphoinositide-3-kinase inhibitor (e.g.,wortmannin or derivatives such as demethoxyviridin, PX-866, LY294002,and LY294002 RGDS (Arg-Gly-Asp-Ser)-conjugated pro-drug SF1126, see,e.g., Maira et al., Biochem. Soc. Trans., 37,265-272 (2009)); NF-kappa-Binhibitor, I-kappa-B kinase inhibitor, mTOR (mammalian target ofrapamycin) inhibitor (e.g., rapamycin, CCI-779, RAD001, or AP23573, seeMaira et al., supra), mitogen activated protein (MAP) kinase inhibitor(e.g., SB-203580 and VX-745, see Brown et al., J. Inflammation, 5:22(2008), or a Janus kinase (JAK) inhibitor.

An antibody or antigen binding fragment also can be conjugated with aliposome. See, for example, the nanoliposomes of Low et al. (Accounts ofChemical Research, 41(1):120-129 (2008)) that are less than 100 nm indiameter and contain a portion of PEGylated lipids (i.e., a lipid linkedto polyethylene glycol (PEG)). Liposomes can be loaded with apharmaceutical agent using methods known in the art.

An antibody or antigen binding fragment described herein can be linkedto a toxin such as Pseudomonas exotoxin A (PE), diphtheria toxin (DT),gelonin, saporin, ricin A, abrin, mistletoe lectin, modeccin, pokeweedantiviral protein (PAP), Bryodin 1, bouganin, or biologically activefragments thereof, to generate an immunotoxin. See, for example,Kreitman, BioDrugs, 23(1):1-13 (2009). PE and DT, and biologicallyactive fragments thereof, are particularly useful. A biologically activefragment of PE can include, for example, amino acids 253-364 and 381-613of PE as described by Hassan et al., J Immunother., 23:473-9 (2000). Abiologically active fragment of DT can include DT388 or DAB389, whichcontain the first 388 or 389 amino acids of DT. See, for example,Chaudhary et al., Biochem Biophys Res Commun., 180:545-51 (1991). Suchimmunotoxins are useful for killing FRβ expressing cells (e.g., any ofthe cancer cells described herein) in vivo or in vitro.

An antibody or antigen binding fragment described herein can be linkedto a detectable moiety. Suitable detectable moieties include, withoutlimitation, radionuclides (e.g., radionuclides used for in vivodiagnostics such as ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁴Cu, ⁶⁷Cu, ²¹²Bi, ¹²³I, ¹³¹I, ²¹¹At,¹⁷⁷Lu, ⁴⁷Sc, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁵³Sm, ¹⁹⁹Au, ^(99m)Tc, ¹¹¹In, ¹²⁴I, ¹⁸F,¹¹C, ¹⁹⁸Au, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹³N, ^(34m)Cl, ³⁸Cl, ^(52m)Mn, ⁵⁵Co, ⁶²Cu,⁶⁸Ga, ⁷²As, ⁷⁶As, ⁷²Se, ⁷³Se, or ⁷⁵Se; or radionuclides useful for invitro experiments such as ¹²⁵I, ³⁵S, ³H, ³²P, ³³P, or ¹⁴C), fluorescentmoieties (e.g., fluorescein, fluorescein isothiocyanate (FITC), PerCP,rhodamine, or phycoerythrin (PE)), luminescent moieties (e.g., Qdot™nanoparticles supplied by the Quantum Dot Corporation, Palo Alto,Calif.), compounds that absorb light of a defined wavelength, or enzymes(e.g., alkaline phosphatase or horseradish peroxidase). Antibodies canbe indirectly labeled by conjugation with biotin then detected withavidin or streptavidin labeled with a molecule described above. Methodsof detecting or quantifying a detectable moiety depend on the nature ofthe moiety and are known in the art. Examples of detectors include,without limitation, x-ray film, radioactivity counters, scintillationcounters, spectrophotometers, colorimeters, fluorometers, luminometers,and densitometers. Methods of attaching the radionuclide atoms (orlarger molecules/chelates containing them) to an antibody or antigenbinding fragment thereof are known in the art and can include incubatingthe antibody or fragment thereof with the radionuclide under conditions(e.g., pH, salt concentration, and/or temperature) which facilitatebinding of the radionuclide atom or radionuclide atom-containingmolecule or chelate to the antibody or antigen binding fragment (see,e.g., U.S. Pat. No. 6,001,329).

Other examples of detectable moieties that can be linked to an antibodyor antigen binding fragment include imaging agents, such as MRI orcomputed tomography (CT) contrast agents. Non-limiting examples of MRIcontrast agents include gadolinium and manganese chelates, iron salts,or gadolinium compounds such as gadodiamide (OMNISCAN™), gadobenic acid(MULTIHANCE™), gadopentetic acid (MAGNEVIST™), gadoteridol (PROHANCE™),gadofosveset (ABLAVAR™) gadoversetamide (OPTIMARK™), or gadoxetic acid(Eovist, known). Non-limiting examples of CT contrast agents includeiodine based agents such as UROGRAFIN™ TELEBRIX™, GASTROGRAFIN™,OMNIPAQUE™, ULTRAVIST™, or VISIPAQUE™.

In some embodiments, a nucleic acid encoding an antibody or an antigenbinding fragment such as an scFv fragment is included in a construct forproducing a chimeric immune receptor. Chimeric immune receptorstypically include an extracellular portion and an intracellular portion,where the extracellular portion is an antigen binding fragment (e.g., ascFv fragment) having binding affinity for human FRβ and theintracellular portion is at least the intracellular domain of asignaling polypeptide such as the CD3 zeta chain, FcRγ chain, or akinase such as a Syk cytoplasmic phosphotyrosine kinase. A chimericimmune receptor further can include at least the intracellular domain ofa costimulatory polypeptide such as the intracellular domain of CD28 orother costimulatory polypeptide such as OX40 (CD134), CD40L, PD-1, or4-1BB (CD137). In some embodiments, a chimeric immune receptor includesan scFv fragment fused to the nonligand binding part of theextracellular and the entire transmembrane and intracellular domains ofCD28, which is fused with the intracellular domain of FcRγ. Constructsencoding chimeric immune receptors can be introduced ex vivo (e.g.,using a retroviral vector) into T cells (e.g., cytotoxic T cells, CD4+ Tcells, or CD8+ T cells) from peripheral lymphocytes of a given patient,and the resulting engineered T cells containing the chimeric receptorcan be re-introduced into the patient. The engineered T cells canproduce at least one cytokine or lymphokine (e.g., IL2, IL3, IL4, IL5,IL6, IL9, IL10, IL12, or IFNγ). Upon binding of the engineered T cellsto FRβ expressing target cells, the engineered T cells are activated andcan kill the FRβ expressing target cells. See, for example, Eshhar in“Therapeutic Antibodies. Handbook of Experimental Pharmacology 181” Y.Chernajovsky, A. Nissim (eds.), 2008; and Pienert et al., Immunotherapy,1(6): 905-912 (2009).

Methods of Using Antibodies

Typically, a human anti-human FRβ mAb or antigen binding fragmentthereof is administered to a mammal such as a human patient that hasbeen diagnosed with an inflammatory disorder or a cancer expressing cellsurface FRβ. In some embodiments, engineered T cells containing achimeric immune receptor (see above section) are administered to thehuman patient. Non-limiting examples of inflammatory disorders includeatherosclerosis, ischemia/reperfusion injury, transplantation rejection,vasculitis such as Wegener's granulomatosus, inflammatoryosteoarthritis, glomerulonephritis, restenosis, systemic sclerosis,fibromyalgia, sarcoidosis, and autoimmune diseases. Non-limitingexamples of autoimmune diseases include rheumatoid arthritis, systemiclupus erythematosus (SLE), ulcerative colitis, psoriasis, Type 1diabetes (insulin-dependent diabetes mellitus), Crohn's disease,multiple sclerosis, and Sjogren's disease. Inflammatory disorders alsocan include obstructive pulmonary diseases such as asthma or chronicobstructive pulmonary disease (COPD).

The antibodies or fragments described herein also can be administered toa subject suspected of having an inflammatory disorder. A subject“suspected of having an inflammatory disorder” is one having one or moresigns of the disorder. Signs of such disorders are well-known to thoseof skill in the art and include, without limitation, redness, swelling(e.g., swollen joints), skin rashes, joint pain, joint pain, loss ofjoint function, fever, chills, fatigue, loss of energy, headaches, lossof appetite, muscle stiffness, insomnia, itchiness, stuffy nose,sneezing, coughing, or one or more neurologic symptoms such as weakness,paresthesias, paralysis, dizziness, seizures, or pain. Signs of diabetesinclude, without limitation, higher than normal frequency of urination,unusual thirst, extreme hunger, unusual weight loss, extreme fatigue,visual problems, and irritability.

Non-limiting examples of cancers expressing cell surface FRβ includemyeloid cancers such as acute myeloid leukemia (AML) or chronic myeloidleukemia (CML), multiple myeloma, or a solid cancer containing FRβexpressing cells such as squamous cell carcinoma of the head and neck,or a malignancy of non-epithelial origin. Treatment of an inflammatorydisorder or cancer can include reducing the severity of the disorder orslowing progression of the disorder.

A human anti-human FRβ mAb, or antigen binding fragment thereof, alsocan be administered prophylactically in subjects at risk for developingan inflammatory disorder to prevent development of symptoms of thedisorder from occurring, delay onset of symptoms, or lessen the severityof subsequently developed disorder symptoms. A subject “at risk ofdeveloping an inflammatory disorder” refers to a subject with a familyhistory of one or more inflammatory disorders (e.g., a geneticpredisposition to one or more inflammatory disorders) or one exposed toone or more inflammation-inducing conditions. For example, a subject canhave been exposed to a viral or bacterial superantigen such as, but notlimited to, Staphylococcal enterotoxins (SEs), a Streptococcus pyogenesexotoxin (SPE), a Staphylococcus aureus toxic shock-syndrome toxin(TSST-1), a Streptococcal mitogenic exotoxin (SME) and a Streptococcalsuperantigen (SSA). An antibody or fragment thereof also can beadministered to deplete activated macrophages from a human subject. Inany case, an amount of the antibody or antigen binding fragment thereofeffective to reduce the number of FRβ positive cells (e.g., macrophagesand monocytes, or cancer cells) in the patient is administered. Thenumber of FRβ positive cells can be determined by doing cell counts orusing a folate-targeted imaging agent.

Methods described herein can include monitoring the patient to, forexample, determine if the disorder is improving with treatment. Anymethod can be used to monitor an inflammatory disorder or cancer. Forexample, for rheumatoid arthritis patients, joint pain and/or stiffness,or bone erosion can be monitored in the patient. For cancer patients,tumor size, cell count, or cancer specific markers can be monitored.

Antibodies or antibody fragments described herein (with or withoutlinked moieties) may be administered by any available route including,but not limited to, oral or parenteral routes of administration such asintravenous, intramuscular, intraperitoneal, subcutaneous, intrathecal,intraarterial, nasal, transdermal (e.g., as a patch), or pulmonaryabsorption. Antibodies or antibody fragments may include a deliveryagent (e.g., a cationic polymer, peptide molecular transporter,surfactant, etc.) as a composition containing a pharmaceuticallyacceptable carrier. As used herein the term “pharmaceutically acceptablecarrier” includes solvents, dispersion media, coatings, antibacterialand antifugal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Supplementaryactive compounds can also be incorporated into pharmaceuticalformulations as described herein.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Solutions or suspensions used forparenteral administration can include the following components: asterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use typicallyinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersion. For intravenous administration,suitable carriers include physiological saline, bacteriostatic water,the polyoxyl castor oil CREMOPHOR™ EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). In all cases, the composition should besterile and should be fluid to the extent that easy syringabilityexists. Pharmaceutical formulations are ideally stable under theconditions of manufacture and storage and should be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.In general, the relevant carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be advantageous to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating theantibody or antibody fragment in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the purified antibody or antibody fragmentinto a sterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions,exemplary methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, theantibody or antibody fragment can be incorporated with excipients andused in the form of tablets, troches, or capsules, e.g., gelatincapsules. Oral compositions can also be prepared using a fluid carrierfor use as a mouthwash. Pharmaceutically compatible binding agents,and/or adjuvant materials can be included as part of the composition.The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. Formulations fororal delivery may advantageously incorporate agents to improve stabilitywithin the gastrointestinal tract and/or to enhance absorption.

For administration by inhalation, the antibody or antibody fragment anda delivery agent are preferably delivered in the form of an aerosolspray from a pressured container or dispenser which contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer. Thepresent disclosure particularly contemplates delivery of thecompositions using a nasal spray, inhaler, or other direct delivery tothe upper and/or lower airway. According to certain embodiments,antibody or antibody fragment and a delivery agent are formulated aslarge porous particles for aerosol administration.

Systemic administration also can be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the purified polypeptide or protein anddelivery agents are formulated into ointments, salves, gels, or creamsas generally known in the art.

In certain embodiments, compositions are prepared with carriers thatwill protect the antibody or antibody fragment against rapid eliminationfrom the body, such as a controlled release formulation, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art. The materials can also be obtainedcommercially from Alza Corporation and Nova Pharmaceuticals, Inc.Liposomal suspensions can also be used as pharmaceutically acceptablecarriers. These can be prepared according to methods known to thoseskilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active antibody or antibodyfragment calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

The antibody or antibody fragment can be administered at variousintervals and over different periods of time as required. Those ofordinary skill in the art will appreciate that certain factors caninfluence the dosage and timing required to effectively treat a subject,including but not limited to the severity of the disease or disorder,previous treatments, the general health and/or age of the subject, andother diseases present. Generally, treatment of a subject with anantibody or antibody antigen-binding fragment as described herein caninclude a single treatment or, in many cases, can include a series oftreatments. It is furthermore understood that appropriate doses maydepend upon the potency of the antibody or antibody fragment and mayoptionally be tailored to the particular recipient, for example, throughadministration of increasing doses until a preselected desired responseis achieved. It is understood that the specific dose level for anyparticular animal subject may depend upon a variety of factors includingthe activity of the specific polypeptide or protein employed, the age,body weight, general health, gender, and diet of the subject, the timeof administration, the route of administration, the rate of excretion,any drug combination, and the degree of expression or activity to bemodulated.

Pharmaceutical formulations as described herein can be included in acontainer, pack, or dispenser together with instructions foradministration.

In some embodiments, an antibody or antigen binding fragment thereof isadministered in combination with one or more pharmaceutical agents(e.g., an anti-inflammatory agent) or antibodies.

Articles of Manufacture

Antibodies or antigen binding fragments described herein thatspecifically bind FRβ can be combined with packaging material and soldas a kit for treating inflammatory disorders, treating a cancerexpressing cell surface FRβ, or depleting activated macrophages from asubject. The articles of manufacture may combine one or more humananti-human FRβ antibodies or fragments thereof. In addition, thearticles of manufacture may further include reagents such as secondaryantibodies, buffers, indicator molecules, solid phases (e.g., beads),pharmaceutical agents, and/or other useful reagents for treatinginflammatory disorders, treating cancer, or depleting activatedmacrophages from a subject. Instructions describing how the variousreagents are effective for treating inflammatory disorders, treatingcancer, or depleting activated macrophages from a subject also may beincluded in such kits.

Certain embodiments of methods and compositions provided herein arefurther illustrated by the following examples. The examples are providedfor illustrative purposes only, and not to be construed as limiting thescope or content of the invention in any way.

EXAMPLES Example 1 Expression of Recombinant Folate Receptor Beta

A nucleic acid encoding a fragment of the human folate receptor beta(FRβ) spanning amino acids 22 to 236 (numbering based on sequence inGenBank Accession No. NP_001107007) was cloned from pcDNA3 tobaculovirus transfer vector pAcGP67 via Sma I and EcoRI sites. FIG. 1contains the amino acid sequence (SEQ ID NO:9) of the FRβ protein.Transfer vector pAcGP67 (BD Biosciences Pharmingen) was co-transfectedwith BaculoGold™ (BD Biosciences Pharmingen) viral DNA into SF9 insectcells according to the manufacturer's instruction.

Recombinant FRβ protein produced from pAcGP67 had four extra residues(ADPG, SEQ ID NO:12) on the N-terminus and five extra histidine residueson the C-terminus. The recombinant FRβ protein was purified fromconditioned medium with a nickel-chelating column, and further purifiedwith a SUPERDEX™ 75 10/300GL gel filtration column in phosphate bufferedsaline (PBS). The purified protein was >95% purity as estimated bySDS-PAGE. The recombinant FRβ retained its function of binding tofolate.

Example 2 Antibody Selection by Phage Display

Purified FRβ was used for panning of a human naïve (not deliberatelyimmunized) Fab phage library. Briefly, 2 μg of FRβ was coated on aMaxisorp plate, which was then incubated with library phage (˜10¹² pfu)for 2 hr. After extensively washing the wells with PBS+0.05% TWEEN20™polysorbate nonionic surfactant (PBST), phage were rescued by incubatingwith exponentially growing TG1 bacteria and helper phage. The panningprocedure was repeated three more times with more stringent conditionsin the latter two rounds, including decreased amounts of FRβ protein andadditional washes with PBST. Three hundred colonies were picked from thelast two rounds of panning and rescued with helper phage.

Two clones were selected and further affinity improved by light chainshuffling in which two sub-libraries were made by replacing each lightchain sequence with the light chain repertoire from the originallibrary. The sub-libraries then were further panned and screened withFRβ protein as described above.

One clone, m923, identified by this method specifically bound to FRβprotein and contained the heavy chain sequence set forth in SEQ ID NO:7and the light chain sequence set forth in SEQ ID NO:8.

The clone with the highest affinity, m909, was further characterized asdescribed below. FIGS. 2A and 2B contain the m909 Fab heavy chainsequence (SEQ ID NO:7) and m909 Fab lamba light chain sequence (SEQ IDNO:17), respectively, with the CDR sequences underlined, frameworkregion sequences italicized, and constant region sequence in lowercase,non-italicized text.

The m909 Fab was expressed in E. coli HB2151 cells. Soluble Fab waspurified from the periplasm using a Ni-chelating column, as the phagemidcarries sequences encoding His6 and FLAG tags at the C-terminus of theFab. A single chain form (scFv) of m909 was made by cloning the VH andVL from the Fab, connected by a linker (GGGGS; SEQ ID NO:13), intopComb3x. Expression and purification of scFv were similar to that of theFab.

The Fab fragment was converted into an IgG1 molecule by subcloning theheavy chain variable region and the light chain into pDR12 to producepDR12-m909. See, Bender et al., Hum Antibodies Hybridomas, 4:74-9(1993). FreeStyle™ 293 cells were transfected with pDR12-m909, and IgG1was secreted into the medium. IgG1 was purified with a protein G column.scFv was purified with a nickel-chelating column. All preparations weredialysed against PBS.

Example 3 Characterization of the m909 Antibody

To characterize the m909 antibody, an ELISA was performed as follows.FRβ, diluted in PBS, was coated on half of the wells of a 96 well plateat 50 ng/well overnight at 4° C. Wells were blocked with 100 μl of 4%milk/PBS (MPBS) for 1 hr at 37° C. For Fab binding kinetics, Fab wastitrated from 3000 nM to 0.038 nM (1:5 serial dilutions) then 50 μl ofdiluted Fab was added to duplicate wells. After 2 hr incubation at 37°C., the wells were washed four times with PBST (PBS+0.05% TWEEN 20™polysorbate nonionic surfactant). Bound Fab was detected by incubatingwith anti-FLAG-horseradish peroxidase (HRP) mAb (1:1000) (Sigma) for 1hr at 37° C. Wells were washed again with PBST. The HRP substrate2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) was added(50 μl/well), and the absorbance at 405 nm was determined. For ELISAwith IgG, a goat anti-human Fc IgG conjugated with HRP was used at1:1000. FIG. 3 is a graph depicting the binding of m909 IgG₁ to FRβ.M909 does not detectably bind to FR alpha (FRI), which shares 70% aminoacid identity with FRβ.

FRβ expression of Chinese hamster ovary (CHO)-K1, CHO cells stablytransfected with human FRβ (CHO-FRβ), KB nasopharyngeal epidermoidcells, and preB L1.2 cells was determined using folate conjugated tofluorescein isothiocyanate (FITC) (FITC-folate) and mouse mAb 94b.CHO-K1 cells did not detectably express cell surface FRβ while theCHO-FRβ cells had high levels of cell surface FRβ. KB and preB L1.2cells had low but detectable levels of cell surface FRβ.

Binding of m909 to cell surface FRβ was determined using FFC on aFACSCalibur (Beckton Dickinson) flow cytometer. Aliquots of cells wereincubated with primary antibody (m909, or isotype controls) at theindicated concentrations (see FIGS. 4 and 5) in 250 μl of folate-freeRPMI+10% FBS for 1 hr on ice. Unbound antibodies were washed away withfolate-free RPMI. Secondary antibody goat anti-human IgG conjugated withFITC (Sigma) was incubated with cells at a concentration of 8 μl/ml for30 min. m909 Fab and scFv were also pre-incubated with mouse anti-His6mAb for 30 min at room temperature, before they were added to cells. Fordetection of Fab or scFv, 1.6 μg/ml of anti-His6 monoclonal antibody(Qiagen) and 8 μl/ml of goat anti-mouse IgG-FITC (Sigma) were incubatedwith cells. Cells were washed and resuspended in PBS+0.5% BSA for FFC ona FACSCalibur (Beckton Dickinson) flow cytometer. FIG. 4 shows that inFRβ+ cells, the monovalent format of the m909 antibody (scFv) did notbind to cell surface FRβ, whereas the bivalent format, sample 5 (scFvcrosslinked by anti-His6 mAb) shows binding. Fab also does not bind toFRβ. FIG. 5 indicates that in FRβ-cells, neither the monovalent format(scFv) nor bivalent format (IgG) of m909 shows detectable binding.

FIGS. 6A and 6B show the concentration-dependent binding of m909 IgG toFRβ+ cells. In FIG. 6A, CHO-FRβ cells (a) were incubated with serialconcentrations of control FITC-Isotype IgG1, and CHO-FRβ cells (b), KBcells (c), and CHO-K1 cells (d) were incubated with serialconcentrations of control FITC-anti-FRβ IgG1. m909 bound to FRβ-positivecell lines. FIG. 6B is a graph depicting affinity of FITC-anti-FRβ IgG1for CHO-FRβ cells. Saturation binding fit gave a dissociation constant(Kd) of 6.393 nM, which is the concentration of free FITC-anti-FRβ IgG1that would half-saturate FRβ on the cell surface.

Example 4 Cell Lysis by Antibody-Dependent Cell-Mediated Cytotoxicity

Peripheral blood mononuclear cells (PBMC) were isolated from healthyhuman donors with Ficoll-Paque Plus (GE Healthcare). Collections ofblood from donors were approved by NCI-Frederick Research Donor Program.The viability of isolated cells was >95%. PBMC were seeded in a 96 wellplate in RPMI+10% FBS at 500,000 cells/well. Cells were incubated at 37°C. and allowed to attach to the plate for 3 hr. Unattached cells wererinsed off by two washes of warm PBS, cells attached in the wells wereused as the effector cells. Target cells (CHO-K1, CHO-FRβ, or preB L1.2cells) were trypsinized and resuspended into single cell suspensions.The target cells were incubated with various concentrations of m909 IgGor control IgG at room temperature for 30 min then added to effectorcells at 10,000 cells/well. The ratio of effector and target cells was50:1. The plate was centrifuged at 300 g×5 min and incubated at 37° C.for 24 hr. Supernatant (100 μl) was transferred to an all-white plateand 100 μl of CYTOTOX-ONE™ lactate dehydrogenase detection reagent(Promega) was added to each well. Lactate dehydrogenase (LDH) releasedfrom lysed cells converted the CytoTox substrate to fluorescentresorufin, which was measured in a fluorometer at the emissionwavelength of 590 nm (excitation wavelength is 530-560 nm). Thepercentage of specific lysis was calculated as the fluorescence of(experimental treatment-effector cell control)/(high control-target cellcontrol)×100%. Fluorescence of target cells alone treated with 1% TRITONX-100™ nonionic surfactant was used as high control. Each treatment wascarried out in 6 replicate wells. Each assay plate included controlwells.

FIGS. 7 and 8 indicate that m909 induced ADCC was correlated with thelevel of surface FRβ expression. In preB L1.2 cells, cell surface FRβexpression was positive but low, and ADCC was detectable atapproximately 5% (see FIG. 7). In CHO-FRβ cells, expression of surfaceFRβ was much higher, and m909 induced approximately 20% of cell lysis(see FIG. 8).

Example 5 Species-Specific Binding of Human Anti-Human FRβ MonoclonalAntibody to FRβ Positive Cells

Live Yersinia enterocolitica bacterial cells were injectedintraperitoneally (IP) into BALB/c mice to recruit peritoneal FRβ⁺macrophages. Three days after the IP injection, peritoneal cells wereremoved and analyzed by flow cytometry using F4/80 macrophage specificantibody. The cells were stained with either (a) 100 nM Folate-FITC inthe absence (solid black histogram) or presence of an excess (10 μM) offree folic acid to competitively occupy FR (filled gray histogram) or(b) 5 nM FITC-anti-human FRβ m909 IgG1 (solid black histogram) or 5 nMcontrol FITC-Isotype IgG1 (filled gray histogram). The percentage ofFRβ⁺ cells within each gate is shown in FIG. 9A. While there wereapproximately 34.23% of FRβ⁺ cells present (see top panel of FIG. 9A),staining with FITC conjugated anti-human FRβ m909 IgG1 detected only0.15% of FRβ⁺ cells present.

Human peripheral blood mononuclear cells (PBMCs) were stained withfolate conjugated to Oregon Green (FOG) and both phycoerythrin(PE)-anti-CD14 and Tricolor-anti-CD16 antibodies. In the experimentsshown in FIG. 9B, the cells were stained with (a) 100 nM FOG in theabsence (solid black histogram) or presence of an excess (10 μM) of freefolic acid to competitively occupy FR (filled gray histogram) or (b) 5nM FITC-anti-FRβ m909 IgG1 (solid black histogram) or 5 nM controlFITC-Isotype IgG1 (filled gray histogram). The percentage of FRβ⁺ cellswithin each gate is shown in FIG. 9B. FITC-anti-FRβ m909 IgG1 boundselectively to CD14^(high), CD16⁻ monocytes.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of treating a patient having aninflammatory disorder, said method comprising administering to saidpatient an amount of an isolated human monoclonal antibody, or anantigen-binding fragment thereof, that specifically binds human folatereceptor beta (FRβ) effective to reduce the number of FRβ positivemacrophages and FRβ positive monocytes in said patient, wherein theantibody or fragment comprises a heavy chain variable region (V_(H))complementarity determining region (CDR) 1 comprising the amino acidsequence set forth in SEQ ID NO:1; a V_(H) CDR2 comprising the aminoacid sequence set forth in SEQ ID NO:2; a V_(H) CDR3 comprising theamino acid sequence set forth in SEQ ID NO:3; a light chain variableregion (V_(L)) CDR1 comprising the amino acid sequence set forth in SEQID NO:4; a V_(L) CDR2 comprising the amino acid sequence set forth inSEQ ID NO:5; and a V_(L) CDR3 comprising the amino acid sequence setforth in SEQ ID NO:6; or wherein the antibody or fragment comprises aV_(H) CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1;a V_(H) CDR2 comprising the amino acid sequence set forth in SEQ IDNO:2; a V_(H) CDR3 comprising the amino acid sequence set forth in SEQID NO:3; a V_(L) CDR1 comprising the amino acid sequence set forth inSEQ ID NO:14; a V_(L) CDR2 comprising the amino acid sequence set forthin SEQ ID NO:15; and a V_(L) CDR3 comprising the amino acid sequence setforth in SEQ ID NO:16.